3-beta-hydroxy-5-alpha-pregnan-20-one for use in medical treatment

Abstract

The present invention provides the steroid compound 3beta-hydroxy-5alpha-pregnan-20-one for use in treatment of Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder, as well as methods for treating said disorders and pharmaceutical compositions for use in treatment of said disorders.

Claims

1. A method of treating Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder, comprising administering a pharmaceutically effective amount of 3beta-hydroxy-5alpha-pregnan-20-one, to a patient in need thereof.

2. The method according to claim 1, wherein the patient suffers from Tourette's syndrome, and 3beta-hydroxy-5alpha-pregnan-20-one is administered in an amount effective to treat the patient's Tourette's syndrome.

3. The method according to claim 1, wherein the patient suffers from obsessive compulsive disorder, and 3beta-hydroxy-5alpha-pregnan-20-one is administered in an amount effective to treat the patient's obsessive-compulsive disorder.

4. The method according to claim 1, wherein the patient suffers from gambling disorder, and 3beta-hydroxy-5alpha-pregnan-20-one is administered in an amount effective to treat the patient's gambling disorder.

5. The method according to claim 1, wherein 3beta-hydroxy-5alpha-pregnan-20-one is administered in combination with at least one additional active compound, selected from the group consisting of serotonin reuptake inhibitors (SSRI), 5alpha-reducase blockers, typical and atypical neuroleptics, antihypertensive agents, tricyclic antidepressants, and opioid antagonists.

6. The method according to claim 5 wherein the serotonin reuptake inhibitors (SSRI) are selected from the group consisting of citalopram, ecitalopram, fluoxetine, sertraline, and fluvoxamine.

7. The method according to claim 5 wherein the 5alpha-reducase blockers are selected from the group consisting of finasteride or dutasteride.

8. The method according to claim 1, wherein the 3beta-hydroxy-5alpha-pregnan-20-one is administered in a pharmaceutical composition comprising 3beta-hydroxy-5alpha-pregnan-20-one and one or more pharmaceutically acceptable carriers, excipients and/or diluents.

9. The method according to claim 5, wherein the typical and atypical neuroleptics are selected from the group consisting of risperdone, ziprasidone, haloperidol, pimozide and fluphenazine.

10. The method according to claim 5, wherein the antihypertensive agents are selected from the group consisting of clonidine and guanfacine.

11. The method according to claim 5, wherein the tricyclic antidepressant is clomipramine.

12. The method according to claim 1, wherein said 3beta-hydroxy-5-alpha-pregnan-20-one is administered intravenously, nasally, buccally, vaginally, rectally, subcutaneously, percutaneously or orally.

13. The method according to claim 1, wherein said 3beta-hydroxy-5-alpha-pregnan-20-one is administered topically, locally, orally, parenterally or pulmonary.

14. The method according to claim 1, wherein the 3beta-hydroxy-5-alpha-pregnan-20-one is in the form of a pharmaceutically acceptable salt, sulphate or ester.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that GABA increased the current response in a concentration-dependent way providing that the receptor alpha1,beta2,gamma2 had a normal and good response.

(2) FIG. 2 shows that THDOC enhanced GABA mediated current response in a concentration-dependent way.

(3) FIG. 3 shows that androstandiol enhanced 30 μM GABA mediated current response at alpha1,beta2,gamma2 human GABA.sub.A receptor sub type.

(4) FIG. 4 shows that THDOC enhanced 3 μM GABA-mediated current in a concentration dependent way at alpha4,beta3,delta human GABA.sub.A receptor sub type.

(5) FIG. 5 shows that androstandiol enhanced 3 μM GABA-mediated current response at alpha4,beta3,delta human GABA.sub.A receptor sub type.

(6) FIG. 6 shows that 3beta-hydroxy-5alpha-pregnan-20-one antagonized the THDOC effect.

(7) In one aspect of the invention, there is provided 3beta-hydroxy-5alpha-pregnan-20-one, for use in treatment of Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder.

(8) In one embodiment of this aspect, said disorder is Tourette's syndrome.

(9) In one embodiment of this aspect, said disorder is obsessive compulsive disorder.

(10) In one embodiment of this aspect, said disorder is gambling disorder.

(11) In one embodiment of this aspect, said 3beta-hydroxy-5alpha-pregnan-20-one is provided in combination with at least one active compound, selected from serotonin reuptake inhibitors (SSRI), including citalopram, ecitalopram, fluoxetine, sertraline, and fluvoxamine; 5alpha-reductase blockers including finasteride or dutasteride; typical and atypical neuroleptics, including risperdone, ziprasidone, haloperidol, pimozide and fluphenazine; antihypertensive agents including clonidine, guanfacine, tricyclic antidepressants, clomipramine and opioid antagonists.

(12) In one embodiment of this aspect, there is provided a pharmaceutical composition comprising 3beta-hydroxy-5alpha-pregnan-20-one, for said use, together with pharmaceutically acceptable carriers, excipients and/or diluents.

(13) In one aspect of the invention, there is provided use of 3beta-hydroxy-5alpha-pregnan-20-one, in the preparation of a medicament useful in the treatment of Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder.

(14) In one embodiment of this aspect, said use is for treatment of Tourette's syndrome.

(15) In one embodiment of this aspect, said use is for treatment of obsessive compulsive disorder.

(16) In one embodiment of this aspect, said use is for treatment of gambling disorder.

(17) In one embodiment of this aspect, said use of 3beta-hydroxy-5alpha-pregnan-20-one is provided in combination with at least one active compound, selected from serotonin reuptake inhibitors (SSRI), including citalopram, ecitalopram, fluoxetine, sertraline, and fluvoxamine; 5alpha-reducase blockers including finasteride or dutasteride; typical and atypical neuroleptics, including risperdone, ziprasidone, haloperidol, pimozide and fluphenazine; antihypertensive agents including clonidine, guanfacine, tricyclic antidepressants, clomipramine and opioid antagonists.

(18) In one aspect of the invention, there is provided a method of treating, preventing or alleviating Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder, comprising administering a pharmaceutically effective amount of 3beta-hydroxy-5alpha-pregnan-20-one, to a patient in need thereof.

(19) In one embodiment of this aspect, said disorder is Tourette's syndrome.

(20) In one embodiment of this aspect, said disorder is obsessive compulsive disorder.

(21) In one embodiment of this aspect, said disorder is gambling disorder.

(22) In one embodiment of this aspect, said 3beta-hydroxy-5alpha-pregnan-20-one is provided in combination with at least one active compound, selected from serotonin reuptake inhibitors (SSRI), including citalopram, ecitalopram, fluoxetine, sertraline, and fluvoxamine; 5alpha-reductase blockers including finasteride or dutasteride; typical and atypical neuroleptics, including risperdone, ziprasidone, haloperidol, pimozide and fluphenazine; antihypertensive agents including clonidine, guanfacine, tricyclic antidepressants, clomipramine and opioid antagonists.

(23) The present invention concerns a method for the treating, preventing or alleviating Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder in human patients described above, according to which method 3beta-hydroxy-5alpha-pregnan-20-one is administered to said patient. Suitable routes of administration are for example the following: intravenous, nasal, buccal, vaginal, rectal, subcutaneous, percutaneous and oral administration. Percutaneous administration, using the substances formulated as a cream, a gel, and an ointment or in the form of slow-release adhesive medicine patches, is another possible form of administration. In any of these or other routes of administration, the formulation of the composition may be adapted or adjusted according to normal pharmacological procedures, comprising the effective pharmaceutical in a chemical form, suitable for the chosen route, together with suitable adjuvants and vehicles, conventionally used and well-known to a person skilled in the art. Suitable, but not limiting, formulations for 3beta-hydroxy-5alpha-pregnan-20-one are provided in WO2011/087441.

(24) Examples of symptoms and conditions caused by the action of 3alpha-hydroxy-5alpha/beta-steroids on the GABA.sub.A receptor are Tourette's syndrome, obsessive compulsive disorder and gambling disorder, tics frequency and type of presentation.

(25) Exacerbation of Tourette symptoms (tics) caused by long time (days) exposure to 3alpha-hydroxy-5alpha/beta-steroids at stress, can according to the present invention, be prevented, alleviated or treated by the administration of 3beta-hydroxy-5alpha-pregnan-20-one to the patient.

(26) One aspect of the invention, addressing a problem afflicting women with Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder, is menstrual cycle linked increased frequency and/or change in tics type difficulties and a method for the treatment in human patients, according to which method 3beta-hydroxy-5alpha-pregnan-20-one is administered to said patient.

(27) In general, the present invention encompasses the use of 3beta-hydroxy-5alpha-pregnan-20-one, either alone or in combination with other pharmaceuticals, such as serotonin reuptake inhibitors (SSRI), including citalopram, ecitalopram, fluoxetine, sertraline, and fluvoxamine; 5alpha-reductase blockers including finasteride or dutasteride; typical and atypical neuroleptics, including risperdone, ziprasidone, haloperidol, pimozide and fluphenazine; antihypertensive agents including clonidine, guanfacine, tricyclic antidepressants, clomipramine and opioid antagonists for the manufacture of a pharmaceutical for the treatment or prevention of any one of the 3alpha-hydroxy-delta 4-5, 5alpha/beta-steroid related or induced disorders described in the specification, and especially one or several of the following disorders: Tourette's syndrome, obsessive compulsive disorder and gambling disorder.

(28) The invention will be described in the following, non-limiting examples.

Example 1

(29) Assay for Testing GABA.sub.A Receptor Effects of 3Beta-Hydroxy-5Alpha-Pregnan-20-One on Human Alpha1,Beta,Gamma and Alpha4,Beta,Delta GABA.sub.A Receptor Subtype.

(30) Aim:

(31) To investigate the effect of 3beta-hydroxy-5alpha-pregnan-20-one on

(32) 1) GABA.sub.A receptor function in the absence and presence of GABA and

(33) 2) in absence and in presence of the positive GABA.sub.A receptor modulating steroids Tetrahydrodeoxycorticosterone (THDOC) and 3alpha-hydroxy-5alpha-androstan-17-ol (3alpha-OH-adiol) by the Dynaflow™ system on HEK-293 cells. In these tests the protocol was optimized to be similar to the physiological conditions in the synaptic cleft.

(34) Cell Culture:

(35) HEK-293 cells, permanently transfected with the human alpha1,beta,gamma and alpha4,beta,delta GABA.sub.A receptor subtypes, were seeded at a density of 3×10.sup.4/25 cm.sup.2 in cellbind culture flask. The transfected cells were used for patch-clamp experiments 3 days after seeding. When using the cells for patch-clamp experiments the cells were washed twice with oxygen-bubbled extracellular (EC) solution (see below). About 5 mL EC was then added and the cells were kept in the incubator for about 15 minutes. After 15 minutes the cells came loose from the bottom of the flask and were separated by carefully sucking couple of times with a Pasteur pipette.

(36) Dynaflow™ system with Resolve chip was used for patch-clamp experiments: The electrophysiological recordings from HEK-293 cells were performed under voltage-clamp conditions using patch-clamp technique and the Dynaflow™ system (Dynaflow Pro II Platform Zeiss Axiovert 25; Cellectricon AB, Sweden) with Resolve chips as application system.

(37) Patch pipettes were pulled from borosilicate glass and polished to a resistance 2-5 MΩ when filled with suitable intracellular solution (IC) (pH was adjusted to 7.2) and immersed in bath solution (extracellular, EC) (pH is adjusted to 7.4). The recordings were made using an Axopatch 200B amplifier, a Digidata 1322A (Axon instruments, Foster city, USA). Data were acquired using the pCLAMP software sampled at 10 kHz, filtered at 2-10 kHz and analyzed with Clampfit (versions 9.0, both from Axon instruments, Foster city, USA). No higher series resistance than 20 MΩ between pipette and cell membrane was accepted. No series resistance compensation was used. The stability of series resistance was monitored repeatedly from the time course of capacitative transients during the experiments. The measured liquid-junction potential between EC and IC was subtracted in all data presented. All experiments were performed at room temperature (21-23° C.).

(38) Steroids and GABA:

(39) GABA was dissolved in EC-solution by ultra sound for about 40 minutes to the concentration of 10 mM in room temperature. All steroids were dissolved to the concentration of 6 mM in DMSO. The DMSO concentration was 0.1% in all end-solutions, including the wash solution (EC) and the solution with GABA alone. End solutions were the solutions added into the wells of the chip.

(40) Electrophysiology:

(41) After compensating for the liquid junction potential a steady holding potential of −17 mV was used in all experiments. In physiological conditions the HEK-293 had a resting potential at −40 mV and a low concentration of chloride ions inside the cell. By using the holding potential of −17 mV and the intracellular solution with low chloride ion concentration the chloride ions flux into the cell when the receptors were activated.

(42) Protocol

(43) GABA Applications:

(44) By using the Dynaflow equipment, it was possible to study transfected HEK-293 during almost physiological conditions. The Dynaflow system allowed application of solutions for as short as 40 ms up to minutes in time. Physiologically, in the synaptic cleft, GABA was released in mM range for about 2 ms. In these experiments we apply GABA±steroid for 40 ms. It was found that in almost all cells, the first GABA application gave a smaller response than the second GABA application. There was no difference in response between the second and the third GABA application. Therefore, the first GABA application was always repeated twice and the second response was used in the analysis.

(45) Washout:

(46) GABA is quite soluble in water and easy to washout from the receptor. The washout time was set to 1 minute after application with GABA solely. Steroids on the other hand were difficult to dissolve in water and also difficult to washout from the receptor. In the experiments, THDOC and 3alpha-OH-adiol were used as the positive GABA.sub.A receptor modulating steroids. With 2 minutes washout time, 200 nM THDOC and 3alpha-OH-adiol was completely washed out as shown by neither an accumulative nor a desensitization effect.

(47) Incubation:

(48) To see the effect of the steroids and to achieve stable results it was found that the steroids had to be incubated on the receptor before application of GABA. Different incubation times were studied to achieve the optimal time for attain stable results and minimize the washout time. Incubation time of 20 seconds showed to be the optimal time for washout time of 2 minutes.

Example 2

(49) Biological Evaluation—HEK-293 Cells, Permanently Transfected with Human Alpha1,Beta,Gamma and Alpha4,Beta,Delta GABA.sub.A Receptor Subtypes and Expressing these GABA.sub.A Receptors.

(50) Cell lines permanently expressing a functional human GABA.sub.A receptor was made in following steps. The GABA.sub.A receptor subunits alpha1, beta2 and gamma2L or alpha4, beta3 and delta including introduced Kozac sequences just before the start codons, were subcloned into mammalian expression vectors containing Geneticin, Hygromycin B, and Zeocin resistance, respectively. A HEK-293 cell line stably expressing the three GABA.sub.A receptor subunits was produced by transfection of the subunits one at a time. The transfection was followed by selection with the appropriate antibiotics, cell separation with the use of subunit specific antibodies, and production of single cell colonies. Produced cell lines were analysed with immunocytochemistry for the three GABA.sub.A receptor subunits, followed by selection of a suitable cell line showing for the GABA.sub.A receptor normal and good reactivity towards GABA, THDOC and 3alpha-OH-adiol. The EC.sub.75 was calculated for THDOC and androstandiol and was used as enhancer of GABA when studying the effect of 1 μM 3beta-hydroxy-5alpha-pregnan-20-one.

(51) Results from tests of GABA in human GABA.sub.A receptor subtype alpha1, beta2, gamma2, permanently expressed in HEK-293 cells are presented in FIG. 1 and FIG. 2. FIG. 1 shows that GABA (1-1000 μM) increased the current response in a concentration-dependent way providing that the receptor alpha1,beta2,gamma2 had a normal and good response. FIG. 2 shows that THDOC (30-1000 nM) enhanced GABA mediated current response in a concentration-dependent way. Control=30 μM GABA, was set to 0 (zero). The figure shows that the receptor alpha1,beta2,gamma2 had a normal and good response.

(52) TABLE-US-00001 TABLE 1 THDOC alone significantly induced current response in absence of GABA at alpha4, beta3, delta receptor. THDOC (μM) Baseline shift Mean ± SEM pA (N; P) 0.03 +5 ± 3.3 pA (N = 9; P = 0.008) 0.1 +15 ± 9.5 pA (N = 9; P = 0.003) 0.3 +12 ± 7.9 pA (N = 9; P = 0.019) 1 +69 ± 35 pA (N = 3; P = 0.018)

(53) FIG. 3 shows that androstandiol (0.3-100 μM) enhanced 30 μM GABA mediated current response at alpha1,beta2,gamma2 human GABA.sub.A receptor sub type. The results in FIG. 3 show surprisingly that androstandiol was substantially less potent (48 times) than THDOC (FIG. 2) in enhancing the effect of GABA on the alpha1,beta2,gamma2 subunit receptor.

(54) TABLE-US-00002 TABLE 2 Androstandiol (8-100 μM) induced current response in absence of GABA at alpha1, beta 2, gamma2 receptor. Androstandiol (μM) effect by itself (Baseline shift) Mean ± SEM pA (N; P) 3 +8 ± 3.6 pA (N = 11; P = 0.004) 8 +13 ± 2.3 pA (N = 10; P = 0.005) 10 +11 ± 1.5 pA (N = 10; P = 0.000) 30 +24 ± 6.8 pA (N = 4; P = 0.001) 100 +43 ± 5.7 pA (N = 4; P = 0.001)

(55) Test of GABA in human GABA-A receptor subtype alpha4, beta3, delta permanently expressed in HEK-293 cells.

(56) The recombinant human alpha4,beta3,delta GABA.sub.A receptor showed expected and normal concentration response curve to GABA (data not shown). Further, 1 μM 3beta-hydroxy-5alpha-pregnan-20-one GABA-mediated current response was without any significant effect on GABA alone at alpha4,beta3,delta GABA.sub.A receptor subtypes (data not shown).

(57) FIG. 4 shows that THDOC (0.01-0.3 μM) enhanced 3 μM GABA-mediated current in a concentration dependent way at alpha4,beta3,delta receptor.

(58) FIG. 5 shows that androstandiol (0.03-10 μM) enhanced 3 μM GABA-mediated current response at alpha4,beta3,delta receptor. The results are surprising since the potency of androstandiol in the alpha4,beta3,delta subunit composition was 10 times higher than in the alpha1,beta2,gamma2 subunit (FIG. 3: 0.38 μM vs. 3.4 μM).

Example 3

(59) Experiments with Application of 3Beta-Hydroxy-5Alpha-Pregnan-20-One on the GABA.sub.A Receptor Subtype Alpha1,Beta2,Gamma2 and GABA.sub.A Receptor Subtype Alpha4,Beta,Delta.

(60) Experiments with application on the GABA.sub.A receptor subtype alpha1,beta2,gamma2 showed (a) the inhibitory effect of 3beta-hydroxy-5alpha-pregnan-20-one on the effect of GABA+3alpha-hydroxy-5alpha-pregnan-21-one (THDOC) application at steady state (phasic effect), and (b) effect of 3beta-hydroxy-5alpha-pregnan-20-one on THDOC induced tonic effect (baseline shift). Steroid effect without GABA, steady state and steroid induced baseline shift (without THDOC and GABA), own effect of 3beta-hydroxy-5alpha-pregnan-20-one.

(61) a) In the presence of 30 μM GABA+200 nM THDOC, a single dose of 1 μM 3beta-hydroxy-5alpha-pregnan-20-one antagonized the THDOC enhanced effect by −22.3±5.3% (p<0.001, n=6). In a separate concentration-dependent experiment, 3beta-hydroxy-5alpha-pregnan-20-one in the concentration interval 0.1-1 μM was tested in the presence of 200 nM THDOC and 30 μM GABA. FIG. 6 shows that 3beta-hydroxy-5alpha-pregnan-20-one antagonized the THDOC effect with max −21±4.6% (p<0.001; n=10) which was reached at 1 μM 3beta-hydroxy-5alpha-pregnan-20-one.
b) The modulatory effect of 1 μM 3beta-hydroxy-5alpha-pregnan-20-one in the presence of 30 μM GABA was positive but did not show any significant effect compared to GABA alone (+10.3±7.8%, NS, n=9, data not shown).

(62) TABLE-US-00003 TABLE 3 Summary of the results at the receptor alpha1, beta2, gamma2. Test I.sub.max/E.sub.max IC.sub.50/EC.sub.50 1-1000 μM GABA 2600 pA 3.6 μM 0.03-1 μM THDOC + 30 +115% 70 nM μM GABA 1 μM 3beta-hydroxy- +10.3 ± 7.8% — 5alpha-pregnan-20-one + (N.S, n = 9) 30 μM GABA 1 μM 3beta-hydroxy-5alpha- −21 ± 4.6% — pregnan-20-one + 30 μM (P < 0.001, n = 10) GABA + 200 nM THDOC 0.1-1 μM 3beta-hydroxy-  −21% 300 nM 5alpha-pregnan-20-one + 30 μM GABA + 200 nM THDOC

(63) Table 4 shows application on a GABA.sub.A receptor subtype alpha4,beta,delta: a) GABA application, Steady state; b) the inhibitory effect of 3beta-hydroxy-5alpha-pregnan-20-one on the effect of GABA+3alpha-hydroxy-5alpha-pregnan-21-one (THDOC) application on a GABA.sub.A receptor subtype alpha4,beta,delta at steady state; c) effect on THDOC induced baseline shift, steroid effect without GABA. The THDOC induced baseline shift (without THDOC and GABA) is shown above in Table 1.

(64) TABLE-US-00004 TABLE 4 Effect of 1 μM 3beta-hydroxy-5alpha-pregnan- 20-one on 3 μM GABA, on THDOC ± 3 μM GABA at alpha4, beta3, delta receptor. The effect of 1 μM 3beta- hydroxy-5alpha-pregnan-20-one on Mean ± SEM % (P; N) a) Control 3 μM GABA response (%) −6.7 ± 3.3% (N = 11, NS) b) 0.1 μM THDOC + 3 μM GABA (%) −26 ± 2.4% (N = 13, P = 0.001) c) 0.1 μM THDOC alone baseline (%) −43 ± 3.5% (N = 11, P = 0.003)

(65) Table 5 shows application on a GABA.sub.A receptor subtype alpha1,beta2,gamma2L: a) the inhibitory effect of 3beta-hydroxy-5alpha-pregnan-20-one on the effect of GABA+3alpha-hydroxy-5alpha-androstan-17-ol (3alpha-OH-adiol) application at steady state (phasic effect); b) effect on 3alpha-OH-adiol induced tonic effect (baseline shift); c) Androstandiol effect without GABA is shown in table 2.

(66) TABLE-US-00005 TABLE 5 Effect of 1 μM 3beta-hydroxy-5alpha-pregnan- 20-one on 8 μM androstandiol ± 30 μM GABA at alpha1, beta2, gamma2. The effect of 1 μM 3beta- hydroxy-5alpha-pregnan-20-one on Mean ± SEM % (P; N) a) 8 μM Androstandiol + 30 μM GABA (%) −30 ± 1.7% (N = 12, P = 0.001) b) 8 μM Androstandiol, baseline shift (%) −33 ± 7.6% (N = 8, P = 0.012)

(67) Maximal modulatory effect of 1 μM 3beta-hydroxy-5alpha-pregnan-20-one in presence of 30 μM GABA alone was not significant (10.3±7.8%, NS, n=9). This shows that 1 μM 3beta-hydroxy-5alpha-pregnan-20-one had no effect of GABA itself, therefore no further studies with 3beta-hydroxy-5alpha-pregnan-20-one and GABA alone was done.

(68) Table 6 shows application on a GABA.sub.A receptor subtype alpha4,beta,delta: a) effect on 3alpha-OH-adiol induced baseline shift; b) the inhibitory effect of 3beta-hydroxy-5alpha-pregnan-20-one on the effect of GABA+3alpha-OH-adiol application on a GABA.sub.A receptor subtype alpha4,beta,delta at steady state; c) Androstandiol effect without GABA is shown below in table 7.

(69) TABLE-US-00006 TABLE 6 Effect of 1 μM 3beta-hydroxy-5alpha-pregnan- 20-one on 3 μM GABA, on androstandiol ± 3 μM GABA and on THDOC + 3 μM GABA at alpha4, beta3, delta. The effect of 1 μM 3beta- hydroxy-5alpha-pregnan-20-one on Mean ± SEM % (P; N) a) 3 μM GABA response (%) −6.7 ± 3.3% (N = 11, NS) b) 0.6 μM Androstandiol + 3 μM GABA (%) −18 ± 2.0% (N = 9, P = 0.008) b) 0.8 μM Androstandiol + 3 μM GABA(%) −22 ± 2.6% (N = 6, P = 0.028)

(70) TABLE-US-00007 TABLE 7 Androstandiol 3 μM and 10 μM significantly induce current response in absence of GABA at alpha4, beta3, delta receptor. Androstandiol (μM) Baselineshift Mean ± SEM pA (N; P) 3 +8 ± 3.6 pA (N = 11; P = 0.004) 10 +13 ± 4.8 pA (N = 12; P = 0.000)

(71) Androstandiol (3 μM and 10 μM) had a significant but minor effect on current response in absence of GABA compare to baseline, current response at EC-solution (Table 7). Androstandiol induced current response was so low that the effect of 1 μM UC1010 on the baseline shift was not possible to study.

(72) Finally, 1 μM 3beta-hydroxy-5alpha-pregnan-20-one had no effect by itself at the alpha4,beta3,delta GABA.sub.A receptor by itself, −0.4±0.3 pA (N=11, NS) compare to current response at EC solution (−0.1±0.3 pA; N=10).

Hypothetical Example 1

(73) There are several ways in which biological effects of treatment Tourette's syndrome, obsessive compulsive disorder and/or gambling disorder could be measured and confirmed. One non-limiting example is the transgenic mouse model described in Nordstrom & Burton 2002 (Nordstrom, E. J. & Burton, F. H. A transgenic model of comorbid Tourette's syndrome and obsessive-compulsive disorder circuitry. Mol. Psychiatry 7, 617-625, 524 (2002)), at least in relation to Tourette's syndrome. In brief, experiments are carried out as follow:

(74) Animals

(75) Male Balb/c mice with heterozygous D1CT-7 mutation (about 3 month-old) weighing approximately 20-30 g. Animals are purchased by Jackson Labs (Bar Harbor, Me.) and genotyped at arrival. Animals are housed in group cages with ad libitum access to food and water. The room is typically maintained at 22° C., on a 12 h: 12 h light/dark cycle from 8 am to 8 pm. Animals are tested during their light cycle between 12 and 4 pm to minimize any potential circadian effects.

(76) Treatment

(77) Treatment is with daily injections of 3beta-hydroxy-5alpha-pregnan-20-one subcutaneously (for instance, but not limited to, as a suspension in MCT oil) or placebo (the vehicle, e.g. MCT oil) in a randomized protocol.

(78) Behavioral Studies

(79) Tic-like manifestations are scored by trained observers blinded to the treatment, according to Nordstrom and Burton (2002). Tic-like manifestations are defined as rapid (<1 second) twitches of the head and/or body.

(80) Statistical Analyses

(81) Non-parametric statistics will be used. The significance threshold could be set at 0.05.

(82) Collection of Blood Samples after 3Beta-Hydroxy-5Alpha-Pregnan-20-One Treatment

(83) A separate set of animals not participating in the behavioral study is treated daily subcutaneously with 3beta-hydroxy-5alpha-pregnan-20-one in appropriate concentration for three days, and blood samples are taken at different time points, such as 2, 4, and 8 h, for determination of plasma concentration of 3beta-hydroxy-5alpha-pregnan-20-one.

CONCLUSIONS

(84) The present invention thus surprisingly provides a possible treatment of Tourette's syndrome, obsessive compulsive disorder and gambling disorder as 3beta-hydroxy-5alpha-pregnan-20-one is active as a GABA.sub.A steroid modulator antagonist and thus can block the enhancement that the GABA.sub.A receptor modulating steroids induce in the main receptor subtypes located in thalamus of patients with Tourette's syndrome, obsessive compulsive disorder and gambling disorder.

(85) 1 μM 3beta-hydroxy-5alpha-pregnan-20-one acted as an antagonist to both androstandiol and to THDOC. 3beta-hydroxy-5alpha-pregnan-20-one significantly reduced the THDOC and androstandiol enhanced GABA mediated effect both at alpha1,beta2,gamma2 and at alpha4,beta3,delta GABA.sub.A receptor subtypes. Further, 1 μM 3beta-hydroxy-5alpha-pregnan-20-one significantly reduced the THDOC enhanced GABA mediated current response without any effect on GABA at alpha4,beta3,delta GABA.sub.A receptor subtype. 1 μM 3beta-hydroxy-5alpha-pregnan-20-one alone had no effect at the alpha4,beta3,delta GABA.sub.A receptor subtype.

(86) Androstandiol enhanced the GABA mediated current response at both alpha1,beta2,gamma2 and alpha4,beta3,delta GABA.sub.A receptor subtypes. However, the EC.sub.50 and E.sub.max differed between the subtypes. At the alpha1,beta2,gamma2 receptor the androstandiol EC.sub.50 was 3.4 μM compared to 0.38 μM on alpha4,beta3,delta. Also, E.sub.max at the alpha1,beta2,gamma2 was 281% compared to Emax=142% at alpha4,beta3,delta. This provides that androstandiol was more efficient but less potent at the alpha1,beta2,gamma2 receptor compare to alpha4,beta3,delta receptor.

(87) THDOC enhanced the GABA mediated current response at alpha4,beta3,delta GABA.sub.A receptor, where the EC.sub.50 was 47 nM and E.sub.max was 297%. This provides that THDOC was more efficient and more potent at alpha4,beta3,delta GABA.sub.A receptor compared to androstandiol.